Compositions containing a urea derivative dye for detecting an analyte and methods for using the same

ABSTRACT

Compositions, reagent test strips, analyte detection systems and kits of the same, as well as methods for their use in the detection of an analyte in a sample, are provided. The subject compositions are characterized by having a positively charged porous matrix and a urea derivative dye on at least one surface of the matrix, where in many preferred embodiments the urea derivative dye is a negatively charged urea derivative dye. In many preferred embodiments, the subject compositions further include at least one additional reagent member of a peroxide producing signal producing system, e.g., an analyte oxidase and/or a peroxidase. The subject compositions, test strips, analyte detection systems and kits find use in the detection of a wide variety of analytes in a sample, such as a physiological sample, e.g., blood or a fraction thereof.

INTRODUCTION FIELD OF THE INVENTION

The field of this invention is analyte detection, particularly reagentsystems for use in analyte detection.

BACKGROUND OF THE INVENTION

Analyte detection in physiological fluids, e.g., blood or blood derivedproducts, is of ever increasing importance to today's society. Analytedetection assays find use in a variety of applications, includingclinical laboratory testing, home testing, etc., where the results ofsuch testing play a prominent role in diagnosis and management in avariety of disease conditions. Analytes of interest include glucose,alcohol, formaldehyde, L-glutamie acid, glycerol, galactose, glycatedproteins, creatinine, ketone body, ascorbic acid, lactic acid, leucine,malic acid, pyruvic acid and uric acid, steroids, etc. In response tothis growing importance of analyte detection, a variety of analytedetection protocols and devices for both clinical and home use have beendeveloped.

Many of the protocols and devices that have been developed to dateemploy a signal producing system to identify the presence of the analyteof interest in a physiological sample, such as blood. One type of signalproducing system that finds use in the detection of a variety ofdifferent analytes is one in which an oxidase or a peroxidase enzymeoxidizes the analyte of interest and produces hydrogen peroxide. Thehydrogen peroxide is then detected by subsequent enzyme-catalyzedreaction with a dye substrate to produce a detectable chromogenicproduct. Dyes useful in such signal producing systems generally yield avisible color signal with high sensitivity.

A variety of highly sensitive dyes are commonly used in peroxideproducing signal producing systems. However, many highly sensitive dyessuch as urea derivative dyes are limited by their stability (e.g., tooxidation, light) in the solid phase and/or in solution.

While compositions containing highly sensitive dyes have been developed,there continues to be a need for the further development of suchcompositions. For example, solid phase compositions that include highlysensitive urea derivative dyes and are resistant to oxidation would beof great interest.

Relevant Literature

Patents of interest include: JP 1118768; JP 9019296; EP 38 205, EP 124287 and EP 251297. See also, Yagi et al. (1986) Biochem. Int.12:367-371.

SUMMARY OF THE INVENTION

Compositions, reagent test strips, analyte detection systems and kits ofthe same, as well as methods for their use in the detection of ananalyte in a sample, are provided. The subject compositions arecharacterized by having a positively charged porous matrix and a ureaderivative dye on at least one surface of the matrix, where in manypreferred embodiments the urea derivative dye is a negatively chargedurea derivative dye. In many preferred embodiments, the subjectcompositions further include at least one additional reagent member of aperoxide producing signal producing system, e.g., an analyte oxidaseand/or a peroxidase. The subject compositions, test strips, analytedetection systems and kits find use in the detection of a wide varietyof analytes in a sample, such as a physiological sample, e.g., blood ora fraction thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 and 2 provide a graphical representation of the UV absorbance ofvarious commercially available dye substrates (1 mM) after reacting withhorseradish peroxidase (1 mg/mL, 549 U/mg) and hydrogen peroxide(50 μM)in pH 7.4 PBS buffer (0.02 M).

FIG. 3 provides a graphical representation of the effects of hydrogenperoxide concentration on DA67 and TOOS-4AP coated ONE-TOUCH® nylonmembranes. TOOS-4AP strips were coated with a 0.02 M PBS, pH 7.4solution containing 5 mM TOOS and 4AP respectively, 1 mg/ml HRP and 1%PVP (360K). The DA-67 strips were coated with 2.5 mM DA-67, 1 mg/ml HRP,1% PVP(360 K) in 0.5 M pH 8.0 phosphate buffer

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Compositions, reagent test strips, analyte detection systems and kits ofthe same, as well as methods for their use in the detection of ananalyte in a sample, are provided. The subject compositions arecharacterized by having a positively charged porous matrix and a ureaderivative dye on at least one surface of the matrix, where in manypreferred embodiments the urea derivative dye is a negatively chargedurea derivative dye. In many preferred embodiments, the subjectcompositions further include at least one additional reagent member of aperoxide producing signal producing system, e.g., an analyte oxidaseand/or a peroxidase. The subject compositions, test strips, analytedetection systems and kits find use in the detection of a wide varietyof analytes in a sample, such as a physiological sample, e.g., blood ora fraction thereof. In further describing the subject invention, thesubject compositions are discussed first, followed by a review ofrepresentative applications in which the compositions find use andsystems and kits that include the subject compositions.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, singular referencesinclude the plural, unless the context clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs.

Compositions and Reagent Test Strips

As summarized above, the subject invention provides compositions for usein detecting a wide variety of analytes in a sample. The compositionsinclude a positively charged porous matrix and a urea derivative dye onat least one surface of the matrix. In many embodiments, however, thecompositions further include at least one additional reagent member of aperoxide producing signal producing system, and typically include aplurality of signal producing system members. The reagent compositionsare dry compositions, such as are typically found in reagent teststrips. In particular, the invention provides strips for assaying for aparticular analyte in whole blood, e.g., glucose, alcohol, glycatedproteins, etc. In the broadest sense, the reagent test strips include apositively charged porous matrix and a peroxide producing signalproducing system present on said matrix, which system includes a ureaderivative dye. In most embodiments, the signal producing system presenton said matrix further includes an analyte oxidizing enzyme.

The above elements are now further described in greater detail.

Positively Charged Porous Matrix

The matrix that is employed in the subject test strips is an inertporous matrix which provides a support for the various members of thesignal producing system, described infra, and has a positive charge. Thematrix is configured to provide a location for application of aphysiological sample, e.g., blood, and detection of the chromogenicproduct produced by the dye of the signal producing system. As such, thematrix is one that is permissive of aqueous fluid flow through it andprovides sufficient void space for the chemical reactions of the signalproducing system to take place. A number of different positively chargedporous matrices have been developed for use in various analyte detectionassays, which matrices may differ in terms of materials, pore sizes,dimensions and the like, where representative matrices include thosedescribed in U.S. Pat. Nos: 55,932,431; 5,874,099; 5,871,767; 5,869,077;5,866,322; 5,834,001; 5,800,829; 5,800,828; 5,798,113; 5,670,381;5,663,054; 5,459,080; 5,459,078; 5,441,894 and 5,212,061; thedisclosures of which are herein incorporated by reference. Thedimensions and porosity of the test strip may vary greatly, where thematrix may or may not have a porosity gradient, e.g., with larger poresnear or at the sample application region and smaller pores at thedetection region. Positively charged membranes can be prepared by usingpositively charged polymers, such as polyamide. Alternatively, suchmembranes can be prepared by various techniques, such as surface coatingusing cationic surfactants or polymers. The coating can be applied bydip coating, chemical treatment, photografting, plasma polymerization,etc. In yet other embodiments, the membrane can be prepared by means ofblending one or more positively charged material with the membraneforming polymer. Examples of positively charged polymers arc polyamide,poly(vinyl pyridine), poly(vinyl imidazole), poly(allylamine),poly(vinyl benzyldimethyl ammonium chloride), polylysine. Examples ofcationic surfactants include those containing primary, secondary andquaternary amino groups. The material may or may not be functionalizedto provide for covalent or noncovalent attachment of the various membersof the signal producing system, described in greater detail infra.

In many embodiments, the matrix is configured as a membrane test pad andis affixed to a solid support, where the support may be a plastic (e.g.,polystyrene, nylon or polyester) or metallic sheet or any other suitablematerial known in the art. Of interest in many embodiments are the teststrip configurations disclosed in U.S. Pat. Nos. 5,972,294; 5,968,836;5,968,760; 5,902,731; 5,846,486; 5,843,692; 5,843,691; 5,789,255;5,780,304; 5,753,452; 5,753,429; 5,736,103; 5,719,034; 5,714,123;383,550; 381,591; 5,620,863; 5,605,837; 5,563,042; 5,526,120; 5,515,170;367,109; 5,453,360; 5,426,032; 5,418,142; 5,306,623; 5,304,468;5,179,005; 5,059,394; 5,049,487; 4,935,346; 4,900,666 and 4,734,360, thedisclosures of which are herein incorporated by reference.

Signal Producing System

In addition to the positively charged porous matrix, the subject teststrips further include one or more members of a signal producing systemwhich produces a detectable product in response to the presence ofanalyte, which detectable product can be used to derive the amount ofanalyte present in the assayed sample. In the subject test strips, theone or more members of the signal producing system are associated, e.g.,covalently or non-covalently attached to, at least a portion of (i.e.,the detection region) the positively charged porous matrix, and in manyembodiments to substantially all of the positively charged porousmatrix.

The signal producing system is an analyte oxidation signal producingsystem. By analyte oxidation signal producing system is meant that ingenerating the detectable signal from which the analyte concentration inthe sample is derived, the analyte is oxidized by a suitable enzyme toproduce an oxidized form of the analyte and a corresponding orproportional amount of hydrogen peroxide. The hydrogen peroxide is thenemployed, in turn, to generate the detectable product from one or moreindicator compounds, where the amount of detectable product produced bythe signal producing system, i.e., the signal, is then related to theamount of analyte in the initial sample. As such, the analyte oxidationsignal producing systems present in the subject test strips are alsocorrectly characterized as hydrogen peroxide based signal producingsystems or peroxide producing signal producing systems.

As indicated above, the hydrogen peroxide based signal producing systemsinclude an enzyme that oxidizes the analyte and produces a correspondingamount of hydrogen peroxide, where by corresponding amount is meant thatthe amount of hydrogen peroxide that is produced is proportional to theamount of analyte present in the sample. The specific nature of thisfirst enzyme necessarily depends on the nature of the analyte beingassayed but is generally an oxidase. As such, the enzyme may be: glucoseoxidase (where the analyte is glucose); cholesterol oxidase (where theanalyte is cholesterol); alcohol oxidase (where the analyte is alcohol);formaldehyde dehydrogenase (where the analyte is formaldehyde),glutamate oxidase (where the analyte is L-glutamic acid), glyceroloxidase (where the analyte is glycerol), galactose oxidase (where theanalyte is galactose), a ketoamine oxidase (where the analyte is aglycated protein, e.g., fructosamine), a 3-hydroxybutyrate debydrogenase(where the analyte is a ketone body), L-ascorbate oxidase (where theanalyte is ascorbic acid), lactate oxidsse (where the analyte is lacticacid), leucine oxidase (where the analyte is leucine), malate oxidase(where the analyte is malic acid), pyruvate oxidase (where the analyteis pyruvic acid), urate oxidase (where the analyte is uric acid oxidase)and the like. Other oxidizing enzymes for use with these and otheranalytes of interest are known to those of skill in the art and may alsobe employed.

The signal producing systems also include an enzyme that catalyzes theconversion of a dye substrate into a detectable product in the presenceof hydrogen peroxide, where the amount of detectable product that isproduced by this reaction is proportional to the amount of hydrogenperoxide that is present. This second enzyme is generally a peroxidase,where suitable peroxidases include: horseradish peroxidase (HRP), soyperoxidase, recombinantly produced peroxidase and synthetic analogshaving peroxidative activity and the like. See e.g., Ci et al. (1990)Analytica Chimica Acta, 233:299-302.

The dye substrates are oxidized by hydrogen peroxide in the presence ofthe peroxidase to produce a product that absorbs light in apredetermined wavelength range, i.e., an indicator dye. Preferably theindicator dye absorbs strongly at a wavelength different from that atwhich the sample or the testing reagent absorbs strongly. The oxidizedform of the indicator may be the colored, faintly-colored, or colorlessfinal product that evidences a change in color of the testing side ofthe membrane. That is to say, the testing reagent can indicate thepresence of an analyte in a sample by a colored area being bleached or,alternatively, by a colorless area developing color.

Dye substrates that are useful in the present invention include ureaderivative dyes. Urea derivative dyes include at least some of thosedisclosed in JP 1118768; JP 9019296; EP 38 205, EP 124 287 and EP251297; the disclosures of which are herein incorporated by reference.The dye substrate is generally a urea derivative, having a negativecharge, where suitable negatively charged urea derivatives include thosebearing a carboxylate group or a sulfonate group. Urea derviative dyesof interest are represented by the following formula:

R¹R²NCONHR³,

wherein

R¹, R² taken together is a N,N-di-substituted aminoaryl; and

R³ is selected from the group consisting of carboxyalkyl,alkoxycarbonyl, alkylcarbonyl, arylsulfonyl, sulfoaryl and carboxyaryl.

The aryl groups of R¹ and R² may be bonded via S to become aphenothiazine derivative type of dye, which is represented by thefollowing formula:

wherein R⁴ and R⁵ are independently selected from NR₂ and OR, where R ishydrogen, (C₁-C₆)-alkyl, (C₁-C₆)alkenyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl; R³ is defined above; and R⁶ and R⁷areindependently selected from hydrogen, (C₁-C₆)-alkyl, (C₁-C₆)-alkenyl,acyl, carboxyl, sulfonyl, nitro, halogen, hydroxyl, (C₁-C₆)-alkoxyl orhydroxy-(C₁-C₆)alkyl.

Alternatively, the aryl groups of R¹ and R² may be bonded via O, to forma phenoxazine derivative type of dye, which is represented by thefollowing formula:

In yet another embodiment, the aryl groups of R¹ and R² is not bonded,which is represented by the following diphenylamine formula:

Exemplary urea derivative dyes include10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine(leuco methylene blue),10-(carboxymethylaminocarbonyl)4,4′-bis(dimethylamino)diphenylamine,10-propionic acid phenothiazine, and salts thereof. In a preferredembodiment, the urea derivative dye is10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine,sodium salt.

Because of the nature of the composition, the urea derivative dyecomponent of the composition is stable, ie., resistant to decompositionby oxidation, as compared to the solution state, especially the aqueoussolution state. As such, the subject compositions can be stored undervarious temperature and humidity conditions with no observable change incolor, i.e., the subject compositions are storage stable. Representativeconditions under which the subject compositions and reagent test stripsare storage stable include in the experimental section, infra Based onthese findings, the subject compositions are stable at temperaturesranging from about at least about −80° C. to 60° C., usually from about−20° C. to 56° C. under humidity ranging from at least about 0% to 80%,usually from about 5% to 20% for periods of time ranging from at leastabout six months to one and a half year, usually from about nine monthsto one year. The subject compositions are more stable than compositionsthat include a neutral or negatively charged porous matrix, e.g., ahydrophilic polysulfone membrane or cellulose filter paper, where themagnitude of this increased stability is at least about one hundredfold.

The subject reagent test strips may be fabricated employing anyconvenient protocol. One convenient protocol is to first contact atleast the test pad portion of the strip with an aqueous solution thatincludes all of the members of the reagent composition that are to beassociated with the test pad in the final reagent test strip except theurea derivative dye. Conveniently, the test pad may be immersed in theaqueous solution, maintained therein for a sufficient period of time andthen dried. The test pad may subsequently be immersed in an organicsolution, e.g., a 70% methanol solution, that includes the ureaderivative dye, maintained therein for a sufficient period of time andthen dried, whereby the test pad of the reagent test strip which hasassociated therewith the composition is produced.

As stated above, the aqueous solution will include various members ofthe composition to be associated with the test pad of the reagent teststrip except the urea derivative dye and the organic solution willinclude the urea derivative dye member where all members are present inamounts sufficient to provide for the desired amounts in the reagentcomposition that is produced on the test pad. As such, the concentrationof the analyte oxidase typically ranges from about 5×10⁻³ to 0.25 mM,usually from about 0.05 to 0.10 mM. Similarly, the peroxidase ranges inconcentration from about 5×10⁻⁴ to 0.125 mM, and usually from about0.005 to 0.05 mM, when present. The concentration of the urea derivativedye typically ranges from about 0.5 to 2 mM, usually from about 0.8 to1.2 mM. Other components that may be present in the aqueous solutionemployed to prepare the reagent test strip include sodium chloride,magnesium chloride, Tris, PSSA, TECTRONIC® 1307 non-ionic surfactant(sold by BASF Corp.), crotein, sucrose, oxamic acid, sodium salt, EDTA,mannitol, polymers such as PVP and PVBTA, and the like. See the examplessection, infra, for a more detailed description of a representativemethod for preparing the subject reagent test strips.

Methods of Analyte Detection

The above described compositions, reagent test strips and signalproducing systems find use in methods of detecting the presence of, andoften the amount of, an analyte in a sample. A variety of differentanalytes may be detected using the subject methods, where representativeanalytes include those described above, e.g., glucose, alcohol,formaldehyde, L-glutarnic acid, glycerol, galactose, glycated proteins,creatinine, ketone body, ascorbic acid, lactic acid, leucine, malicacid, pyruvic acid, uric acid, and steroids, etc. While in principle,the subject methods may be used to determine the presence, and oftenconcentration, of an analyte in a variety of different physiologicalsamples, such as urine, tears, saliva, and the like, they areparticularly suited for use in determining the concentration of ananalyte in blood or blood fractions, e.g., blood derived samples, andmore particularly, in whole blood.

An important feature of the subject methods is that use of the subjectsignal producing systems that include a urea derivative dye provides forthe highly sensitive detection of hydrogen peroxide. As such, hydrogenperoxide may be detected at submillimolar concentrations using thesubject stable dry reagent formats, e.g., test strips, where bysubmillimolar concentration is typically meant concentrations rangingfrom 0.010 to 1 mM, usually from about 0.050 to 0.8 mM. Use of thesubject signal producing systems that include a urea derivative dyeprovides for more sensitive detection of hydrogen peroxide as comparedto signal producing systems that include a dye substrate other than aurea derivative dye, e.g.,N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, 4-aminoantipyrine(see FIG. 3).

In the subject methods, the sample and the signal producing systemincluding a urea derivative dye are combined into a reaction mixture,e.g., by applying the sample to the test strip, the reaction is allowedto proceed for a sufficient period to time to generate a signalindicative of the presence (and often the amount) of analyte in thesample, and the resultant signal is detected and related to the presence(and often the amount) of analyte in the sample. The subject methods arenow discussed further in terms of methods in which a reagent test stripis employed.

In practicing the subject methods, the first step is to apply a quantityof the physiological sample to the test strip, where the test strip isdescribed supra. The amount of physiological sample, e.g., blood, thatis applied to the test strip may vary, but generally ranges from about 2μL to 40 μL, usually from about 5 μL to 20 μL. Because of the nature ofthe subject test strip, the blood sample size that is applied to thetest strip may be relatively small, ranging in size from about 2 μL to40 μL, usually from about 5 μL to 20 μL. Where blood is thephysiological sample, blood samples of a variety of differenthematocrits may be assayed with the subject methods, where thehematocrit may range from about 20% to 65%, usually from about 25% to60%.

Following application of the sample to the test strip, the sample isallowed to react with the members of the signal producing system toproduce a detectable product that is present in an amount proportionalto the initial amount of the analyte of interest present in the sample.The amount of detectable product, i.e., the signal produced by thesignal producing system, is then determined and related to the amount ofanalyte in the initial sample.

Analyte Detection Systems

Analyte detection systems useful for practicing the subject methodsinclude a reagent test strip and an automated instrument. In suchsystems, a physiological sample is applied to the test strip asdescribed above and the signal produced by the signal producing systemis detected and related to the presence (and often the amount) ofanalyte in the sample by the automated instrument. The above describedreaction, detection and relation steps, and instruments for practicingthe same, are further described in U.S. Pat. Nos. 4,734,360; 4,900,666;4,935,346; 5,059,394; 5,304,468; 5,306,623; 5,418,142; 5,426,032;5,515,170; 5,526,120; 5,563,042; 5,620,863; 5,753,429; 5,573,452;5,780,304; 5,789,255; 5,843,691; 5,846,486; 5,902,731; 5,968,836 and5,972,294; the disclosures of which are herein incorporated byreference. In the relation step, the derived analyte concentration takesinto account the constant contribution of competing reactions to theobserved signal, e.g., by calibrating the instrument accordingly.

The subject analyte detection systems include enzyme immunoassay systemsfor analyte determination, where such systems include multiple teststrips forming a strip plate that includes a positively charged porousmatrix and a urea derivative dye on at least one surface of the matrix,e.g., an enzyme-linked immunosorbent assay (ELISA) where the enzyme is aperoxidase and the strip plate further includes an analyte-specificantibody on at least one surface of the matrix.

Kits

Also provided by the subject invention are kits for use in practicingthe subject methods. The kits of the subject invention include a reagenttest strip that includes a peroxide producing signal producing system,as described above, and at least one of a means for obtaining saidphysiological sample, e.g., a lance for sticking a finger, a lanceactuation means, and the like, and an analyte standard, e.g., an analytecontrol solution that contains a standardized concentration of analyte.In certain embodiments, the kits also include an automated instrument,as described above, for detecting the amount of product produced on thestrip following sample application and relating the detected product tothe presence (and often the amount) of analyte in the sample. Finally,the kits include instructions for using the subject kit components inthe determination of an analyte concentration in a physiological sample.These instructions may be present on one or more of the packaging, alabel insert, containers present in the kits, and the like.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Screening of Commercially Available Dye Substrates

The stock solutions of various dyes listed below were prepared in eitherwater or water/0.2M citrate or water/0.2M citrate/ethanol mixture. Theconcentrations of dye stock solutions ranged from 8 mM to 50 mM. A 10mg/ml HRP stock solution was also prepared in 0.02 M PBS, pH 7.4solution. Dye/HRP solution was then prepared by mixing the proper volumeof dye stock solution, 10 mg/ml HRP stock solution and 0.02M PBS bufferto the final concentration of [HRP]=1 mg/ml and [dye]=1 mM. 200 μl ofthe thus prepared dye/HRP was then added to microtiter plate wells,followed by 20 μl 500 mM H₂O₂ solution. The absorbance of the solutionwas taken by microtiter plate reader after the color development reachedsteady state (in less 2 min.)

Dye Substrate Abbreviation 10-(carboxymethylaminocarbonyl)-3,7- DA-67,WAKO's bis(dimethylamino)phenothiazine, sodium saltN-(carboxymethylaminocarbonyl)-4,4′- DA-64bis(dimethylamino)diphenylamine, sodium saltN-ethyl-N-(2-hydroxy-3-sulfopropyl)-3- ADOS methoxyanilineN-ethyl-N-(3-sulfopropyl-3-methoxyaniline ADPSN-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline ALOSN-ethyl-N-(3-sulfopropyl)aniline ALPSN-ethyl-N-(3-sulfopropyl-3-methylaniline TOPSN-ethyl-N-(2-hydroxy-3-sulfopropyl)-3- TOOS methylaniline, sodium salt3,3′,5,5′-tetramethylbenzidine, dihydrochloride TMBZ-HClN(3-sulfopropyl)-3,3′,5,5′-tetramethylbenzidine TMBZ-PS2,2′-azinobis(3-ethylbenzothiazolin-6-sulfonic acid) ABTSN-ethyl-N-3-sulfopropyl)-3,5-dimethylaniline MAPSbis(4-[N-ethyl-N-(3′-sulfopropyl)-amino-2,6- Bis-MAPS-C2bis(4-[N-propyl-N-(3′-sulfopropyl)-amino-2,6- Bis-MAPS-C3bis(4-[N-butyl-N-(3′-sulfopropyl)-amino-2,6- Bis-MAPS-C4N,N-bis(2-hydroxy-3-sulfopropyl)tolidine SATN,N-bis(4-sulfobutyl)-3,5-dimethylaniline MADBN-ethyl-N-2-hydroxy-3-sulfopropyl)-3,5 MAOS dimethylaniline

Example 2 Preparation of Test Strips Coated with a Peroxide ProducingSignal Producing System Including DA-67

A ONE-TOUCH® Nylon membrane was cut into strips of ¼″ wide and 12″ long,and the membrane strip was first coated with the A dip solution and thenwith the B dip solution (ingredient and concentration specified in thefollowing table). After each coating, the membrane was dried in hot airoven for 10 minutes at 55° C. The coated membrane was stuck to a 2″×12″MELINEX® support (a polyester film or sheet made by Du Pont) which has a5 mm diameter circular hole opening every quarter inch along its lengthfor color measurement. The MELINEX® support also has two stripes of ⅜″wide adhesive printed on top and bottom of holes. The membrane waslaminated in the way that it completely covers the holes on the MELINEX®support. A layer of sample-spreading POREX™ material (a porous highdensity polyethylene material made by Porex Corp.) (1″×12″) was furtherlaminated on top of membrane by adhesive printed on MELINEX® support.The whole assembly was then cut into ¼″ wide strips.

Ingredient Concentration A dip formulation Ketoamine oxidase 206U/mlHorseradish peroxidase 1 mg/ml Mannitol 4% Poly(vinylpyrrolidone) (PVP),MW = 360K 1% EDTA 5 mM buffer 0.1M sodium phosphate pH 7.5 B dipformulation DA-67 1.5 mM solvent 70% methanol

Example 3 Detection of Hydrogen Peroxide

To demonstrate the feasibility of color development on strips coatedwith stabilized DA-67 dye, a ONE-TOUCH® Nylon membrane was coated with asolution containing 2.5 mM DA-67, 1 mg/ml HRP and 1% PVP (MW=360K) in0.5M pH 8.0 phosphate buffer. The membrane was then stuck to MELINEX®support, covered with POREX® material and cut into testing strips asdescribed in example 2. Ten μL volumes of various concentrations of H₂O₂solutions were dropped on the strips respectively, and the colorformation was monitored using a MACBETH REFLECTOMETER™ (an instrumentfor measuring color by means of reflectance spectroscopy made byGretagMacbeth Corp.). To compare the detection sensitivity of DA-67 dyewith that of the most commonly used dye for H₂O₂, TOOS-4AP, a ONE-TOUCH®Nylon membrane was coated with a solution containing 5 mM TOOS, 5 mM4AP, 1 mg/ml HRP and 1% PVP (MW=360K) in 0.02M PBS, pH 7.4. The membranewas then used to prepare test strips as described in example 2 andtested with H₂O₂ by the way DA-67 coated strips was tested. The resultswere plotted in FIG. 3.

It is evident from the above results and discussion that the subjectinvention provides for the significant stabilization of a highlysensitive urea derivative dye member of a hydrogen peroxide based signalproducing system useful for analyte detection. As such, the subjectinvention provides for more sensitive analyte detection, as compared tocertain prior art compositions and methods, e.g., ones that rely on dyesubstrates other than urea derivative dyes, and represents a significantcontribution to the art.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference. The citation of any publication is for its disclosure priorto the filing date and should not be construed as an admission that thepresent invention is not entitled to antedate such publication by virtueof prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

What is claimed is:
 1. A storage stable composition of mattercomprising: a positively charged porous matrix comprising nylon; and aurea derivative dye on at least one surface of said matrix, wherein saidurea derivative dye is10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine or asalt thereof; wherein said composition is stable for at least about sixmonths at temperatures ranging from at least about −80° C. to 60° C.under humidity ranging from at least about 0% to 20%.
 2. The compositionaccording to claim 1, wherein said urea derivative dye is a member of aperoxide producing signal producing system present on said matrix. 3.The composition according to claim 2, wherein said composition furthercomprises at least one additional reagent member of a peroxide producingsignal producing system.
 4. The composition according to claim 3,wherein said at least one additional reagent member is an analyteoxidase.
 5. The composition according to claim 3, wherein said at leastone additional reagent member is a peroxidase.
 6. The compositionaccording to claim 5, wherein said peroxidase is horseradish peroxidase.7. A storage stable reagent test strip for use in detecting the presenceor determining the concentration of an analyte in a physiologicalsample, said strip comprising: a positively charged porous matrixcomprising nylon; and a peroxide producing signal producing systempresent on said matrix, wherein said peroxide producing signal producingsystem includes10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine or asalt thereof, wherein said test strip is stable for at least about sixmonths at temperatures ranging from at least about −80° C. to 60° C.under humidity ranging from at least about 0% to 20%.
 8. The test stripaccording to claim 7, wherein said peroxide producing signal producingsystem comprises an analyte oxidase.
 9. The test strip according toclaim 7, wherein said peroxide producing signal producing systemcomprises a peroxidase.
 10. The test strip according to claim 9, whereinsaid peroxidase is horseradish peroxidase.
 11. An analyte detection ormeasurement system comprising: (a) a storage stable reagent test stripcomprising: (i) a positively charged porous matrix comprising nylon; and(ii) a peroxide producing signal producing system present on saidmatrix, wherein said peroxide producing signal producing system includes10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine or asalt thereof; and (b) an automated instrument, wherein said test stripis stable for at least about six months at temperatures ranging from atleast about −80° C. to 60° C. under humidity ranging from at least about0% to 20%.
 12. A method for detecting the presence or determining theconcentration of an analyte in a sample, said method comprising: (a)applying said physiological sample to a storage stable reagent teststrip comprising: (i) a positively charged porous matrix comprisingnylon; and (ii) a peroxide producing signal producing system present onsaid matrix, wherein said peroxide producing signal producing systemincludes10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine or asalt thereof, wherein said test strip is stable for at least about sixmonths at temperatures ranging from at least about −80° C. to 60° C.under humidity ranging from at least about 0% to 20%; (b) detecting asignal produced by said signal producing system; and (c) relating saiddetected signal to the presence or concentration of said analyte in saidphysiological sample.
 13. The method according to claim 12, wherein saidanalyte is selected from the group consisting of glucose, cholesterol,alcohol, formaldehyde, L-glutamic acid, glycerol, galactose, glycatedproteins, creatinine, ketone body, ascorbic acid, lactic acid, leucine,malic acid, pyruvic acid and uric acid.
 14. The method according toclaim 12, wherein said sample is whole blood or a derivative thereof.15. The method according to claim 12, wherein said detecting andrelating steps are carried out by an automated instrument.
 16. A kit foruse in determining the concentration of an analyte in a physiologicalsample, said kit comprising: (a) a storage stable reagent test stripcomprising: (i) a positively charged porous matrix comprising nylon; and(ii) a peroxide producing signal producing system present on saidmatrix, wherein said peroxide producing signal producing system includes10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine or asalt thereof, wherein said test strip is stable for at least about sixmonths at temperatures ranging from at least about −80° C. to 60° C.under humidity ranging from at least about 0% to 20%; and (b) at leastone of: (i) a means for obtaining said physiological sample and (ii) ananalyte standard.
 17. The kit according to claim 16, wherein said meansfor obtaining said physiological sample is a lance.
 18. The kitaccording to claim 16, wherein said analyte standard comprises astandardized concentration of a known reagent.
 19. The kit according toclaim 16, wherein said kit comprises a means for obtaining saidphysiological sample and an analyte standard.